Bottom Line:
Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators.Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression.Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls.

ABSTRACTSarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.

fig7: Effects of PMA/I and merozoite stimulation on peripheral leukocyte proliferation as detected using CFSE. Lymphocytes from control and S. neurona-infect horses were incubated with CFSE and cultured with (a) PMA/I or (b) unstimulated for 72 hrs at 37°C, 5% CO2. Cells were washed and stained for leukocyte markers, and proliferation based on number of divisions was determined, (c) PMA/I stimulated, and (d) unstimulated.

Mentions:
Samples were gated on live versus dying/apoptotic gates, and then cell divisions were marked (Figure 7, Table 3) [23]. For each time point, stimulation, and immune cell subset, data were gated and statistically analyzed based on quantitative differences in number of proliferations present per sample. The significant differences are presented in Table 3 to provide the pattern of response. CD4+ cells (days 2, 7, 21, and 56) and CD8+ cells (days 2, 21, and 28) from S. neurona-infected horses had increased numbers of divisions/proliferation when stimulated with PMA/I. When cultured with live merozoites, CD4+ cells from the S. neurona-infected horses had decreased cell divisions/proliferation on day 28, but increased divisions/proliferation on day 42. CD8+ cells had decreased divisions/proliferation in infected horses at days 28, 56, and 70, suggesting a limited ability of leukocytes to respond to merozoites. Due to a consistently limited cell sample recovery after 72 hrs of culture, the B cells and monocytes were not evaluated.

fig7: Effects of PMA/I and merozoite stimulation on peripheral leukocyte proliferation as detected using CFSE. Lymphocytes from control and S. neurona-infect horses were incubated with CFSE and cultured with (a) PMA/I or (b) unstimulated for 72 hrs at 37°C, 5% CO2. Cells were washed and stained for leukocyte markers, and proliferation based on number of divisions was determined, (c) PMA/I stimulated, and (d) unstimulated.

Mentions:
Samples were gated on live versus dying/apoptotic gates, and then cell divisions were marked (Figure 7, Table 3) [23]. For each time point, stimulation, and immune cell subset, data were gated and statistically analyzed based on quantitative differences in number of proliferations present per sample. The significant differences are presented in Table 3 to provide the pattern of response. CD4+ cells (days 2, 7, 21, and 56) and CD8+ cells (days 2, 21, and 28) from S. neurona-infected horses had increased numbers of divisions/proliferation when stimulated with PMA/I. When cultured with live merozoites, CD4+ cells from the S. neurona-infected horses had decreased cell divisions/proliferation on day 28, but increased divisions/proliferation on day 42. CD8+ cells had decreased divisions/proliferation in infected horses at days 28, 56, and 70, suggesting a limited ability of leukocytes to respond to merozoites. Due to a consistently limited cell sample recovery after 72 hrs of culture, the B cells and monocytes were not evaluated.

Bottom Line:
Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators.Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression.Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls.

ABSTRACTSarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.